This invention relates to a cDNA coding for a rat CNP (rat C-type natriuretic peptide which is hereunder abbreviated as rCNP), as well as the rCNP precursor protein (prepro rCNP) that is encoded by said cDNA.
In recent years, various peptides collectively referred to as "natriuretic peptides" (NP) have been discovered from the atria and brains of various animals. Today those NPs can be classified into three types, A-type natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), depending on similarities in the primary amino acid sequence and the structure of their precursors. Among the three types, ANP and BNP were first isolated and identified from the atrium and brain and hence are sometimes called "atrial natriuretic peptide" and "brain natriuretic peptide", respectively (Matsuo, H. and Nakazato, H., Endocrinol. Metab. Clin. North Am., 16, 43, 1987; Sudoh, T. et al., Nature, 332, 78, 1988). However, it is known today that ANP occurs not only in the atrium but also in the brain and that BNP occurs not only in the brain but also in the atrium. Further, both ANP and BNP exhibit significant natriuretic and hypotensive actions, so it has become clear that each of these peptides works not only as a hormone to be secreted from the atrium into blood but as a nerve transmitter in the brain, in either case helping regulate the homeostatic balance of body fluid volume and blood pressure in mammals.
The CNP has very recently been isolated and identified from porcine brain by Sudoh et al. as a third type of NP that is assignable to neither ANP nor BNP (Sudoh, T. et al., Biochem. Biophys. Res. Commun., 168, 863, 1990). The first discovered CNP consisted of 22 amino acid residues (this peptide is hereunder abbreviated as "pCNP-22"). Like ANP and BNP, CNP contained two cysteine residues which formed an intramolecular disulfide bond, producing a ring structure composed of 17 amino acid residues. Further, the primary amino acid sequence forming that ring structure in pCNP-22 was found to be highly homologous to that in ANP and BNP.
However, pCNP-22 differs from ANP and BNP in that the latter have several amino acid residues additionally attached to the C-terminus of the ring structure whereas no such "tail structure" is present in pCNP-22. In other words, the C-terminus of pCNP-22 is terminated with a cysteine residue. On the basis of these facts, it has been found that the structure of pCNP-22 is similar to but clearly distinguishable from those of ANP and BNP. In addition, pCNP-22 exhibited natriuretic and hypotensive actions and it was also found to display a higher activity than ANP and BNP in relaxant activity tests on chick rectal samples. From these observations, pCNP-22 was found to be an NP assignable to a new type, the peptides of which were named "CNPs".
Following pCNP-22, a second peptide assignable as CNP was isolated and identified from porcine brain by the present inventors. The peptide was found to consist of 53 amino acid residues with pCNP-22 present at the C-terminus (this peptide is hereunder abbreviated as pCNP-53). In other words, pCNP-53 was found to be a peptide that had 31 more amino acid residues attached to the N-terminus of pCNP-22. Interestingly enough, pCNP-53 has been found to occur in a greater amount than pCNP-22 in porcine brain (commonly assigned Japanese Patent Application No. 186582/1990).
Another very recent study succeeded in identifying the structure of the precursor of pCNP-22 and pCNP-53 by gene analysis and this helped unravel the mechanism behind the biosynthesis of those peptides (commonly assigned Japanese Patent Application No. 186583/1990). The present inventors isolated and identified a porcine chromosomal gene and cDNA coding for pCNP-22 and pCNP-53; by their analysis, the structure of the porcine CNP precursor protein (prepro pCNP) was unravelled; at the same time, it was found that each of pCNP-22 and pCNP-53 was first translated from mRNA as a prepro pCNP composed of 126 amino acid residues and that the signal peptide present in the N-terminal region of the precursor protein was then cleaved in the process of secretion to be converted to pro pCNP, which was further cleaved specifically with processing enzymes to be converted to pCNP-53 or pCNP-22 as appropriate. On the basis of these observations, it has been found that as in the case of ANP and BNP, both pCNP-22 and pCNP-53 are secretory peptides that are biosynthesized from a common precursor protein (prepro pCNP).
However, in sharp contrast with the peptides assignable to ANP and BNP which have been found by previous studies to work not only as hormones to be secreted from the atrium into blood but also as nerve transmitters in the brain, thereby regulating the homeostatic balance of body fluid volume and blood pressure, CNP remains unclear in many points as regards details of its distribution in vivo and physiological actions.
As for ANP and BNP, their structures have been established in rats and the physiological actions in rats of rat ANP and BNP have also been unravelled, with the evaluation of their pharmaceutical efficacy being conducted with care being taken to eliminate possible effects due to differences in animal species. However, as of today, the structure of CNP has not been identified in rats.